The proposed research relates to the molecular basis of electrical excitability in nerve and muscle cells. The ionic channels responsible for this excitability have been the focus of much theoretical and experimental work in the past 30 years. The proposed project will use new experimental and analytical techniques to test two major hypotheses about ion permeation through channels and the mechanism of channel gating. I. To what extent does the acetylcholine receptor channel behave (as commonly pictured) as a rigid pore? Thermal fluctuations in the structure of soluble proteins is a topic of considerable interest; in some cases these fluctuations have been shown to also have considerable physiological importance. Using the patch clamp technique it is now possible to resolve the small fluctuations in ion flow that would result from thermal fluctuations in the structure of channel proteins, as well as the "shot noise" that results from the transport of individual ions. In the proposed project, recordings from acetylcholine receptor (AChR) channels will be analyzed and compared with recordings from a model system (gramicidin channels in artifical and muscle-cell membranes) in order to determine the origin and characteristics of the fluctuations in the current through AChR channels. II. What steps determine the rate of activation and inactivation in the voltage-gated sodium channel? Results presented in a recent papr by Aldrich, Corey and Stevens (1983) show, on the basis of single channel measurements in neuroblastoma cells, that the inactivation step in sodium channel gating is intrinsically more rapid than activation. This result is the opposite of commonly-held views of sodium channel function, and it is also in apparent conflict with results from other nerve preparations. A strong test is proposed for a central observation of Aldrich et al, namely that sodium channels open only once during a depolarization. Because the test uses fluctuation analysis of whole-cell currents, it can be performed over a wider potential range than the single channel analysis used by Aldrich et al.